Properties of nitracrine-nucleic acids complexes in selected enzymatic systems

A C T A U N I V E R S I T A T I S L O D Z I E N S I S FOLIA BIOCHIMICA ET BIOPHYSICA 6, 1988

E wa C i e si e ls k a, M a r i o l a P i e s t r z e n i e w i c z , W ł a d y s ł a w Ga łasi ńs ki , M a r e k G n i a z d o w s k i

P R O P E R T I E S O F N I T R A C R I N E - N U C L E I C A C I D S C OM P L EX E S IN S E L E C T E D E NZ Y M A T I C SY ST E MS *

Some properties of the covalent complexes, of the anticancer drug - nitracrine ( l-nitro-9-(3,3-N,N-dimethylaminopropylamino) acridine, Le- dakrin, C-283, NAC) with nucleic acids have been tested in DNA or RNA- -dependent enzymatic systems.

NAC-DNA complexes exhibit decreased template activity in an RNA synthesis in vitro system. They show an increased resistance to DN-ase I and DN-ase Sj and to acid hydrolysis. Binding of NAC to tRNA decrea-ses aminoacylation of the latter. On the other hand, such aminoacyl- -tRNA does not loose activity in the protein synthesis in vitro. Preli-minary experiments on the chromatographic resolution of the hydrolyzed complexes are presented here.

A n a n t i c a n c e r drug, n i t r a c r i n e (l - n i t r o - 9 - ( 3,3 - N , N- d i m et h yl a - m i n o - p r o p y lamino) ac ri di ne, NAC, Le da kr in , C-283) in h ib i ts DNA, RNA, a nd to a lo we r e x t e n t p r o t e i n s y n t h es i s e i t h e r in v iv o or in the ce l l c u lt u r e (see [4] for rev iew) . In th e p r es e n c e of su lfhy - d r yl c o m p o u n d s s e ve r al b i o l o g i c a l l y a ct i v e 1 n i t r o a c r id i n e d e r i v a -ti v es f or m c o v a l e n t c o m p l e x e s w i t h DNA, R N A a nd pr ot ei ns . A stru c- t u r e - a c t i v i t y r e l a t i o n s h i p e x h i b i t e d b y the n i t r o a c r i d i n e s b ot h in th is r e a c t i o n an d in t he b i o l o g i c a l e x p e r i m e n t s p r o m p t e d us to u se the t h i o l - d e p e n d e n t s y s t e m as a m o d e l of the d r u g fa te in the cell. It h as b e e n f o un d th at c o m p l e x e s of N A C w i t h D N A s ho w c o n s i d e r a b l y lo w er t e m p l a t e a c t i v i t y in th e R N A s y nt h es i s in v it r o s y st e m [5, 6, 12, 13]. A s e n s i t i v i t y of N A C - D N A c o m p l e x e s to n u c l e as e s and

the e ff e ct of the d ru g b i nd i n g to t RN A on t RN A f u n ct i on w e re a s -s ay e d in the e xp e r i m en t s r e p o rt e d here. T h e y e n a b l e d a fu rth er c h a r a c t e r i z a t i o n of the N A C - D N A c o mp l e x es an d a p p r o a c h i n g other p o s s i bl e m o l e c u l a r m e c h a n i s m s of the d r u g action.

MATERIALS AND METHODS

9 [ 14 C] NAC, the u n l a b e l l e d d r u g a nd s yn t he t ic p o l y n u c l e o ti d e s w e re the s ame as d e s c r i b e d b e f o re [3, 11]. Y e a st t R NA i s ola te d a c co r d i ng to [15], p a n c r e at i c d e o x y r i b o n u c l e a s e I (E.C. 3.1.21.1). D NA (Wo rthi ngto n, U S A ) , d e o x y r i b o n u c l e a s e S^ f rom A s p er g il l us o ry z ae (E.C. 3.1.30.1, Ca lb io ch em , USA) w e re used. O th e r a n a l y -ti ca l g ra d e c h e m i c al s w e re p u r c h a s e d f r om Cefarm, Poland. C o m p l e x es of N A C w i t h DNA, t RN A or s y nt h et i c p o l y n uc l e o t i d es w e re f o rm e d in the p r e s e n ce of d i t h i ot h re i to l , p u r i f i e d a n d their s t o i ch i o m e t r y w as e s t im a t e d as d e s cr i b e d b ef o re [11]. A m i n o a c y l a t i o n of t R NA w a s a s s a y ed a c c o r d i ng to M an s a nd No- ve ll i [8] u s in g y ea s t t RN A as a c o n tr o l a nd an e q u i v a l e nt a mo un t of N A C - t R N A co mple x, U - [ 14C] a mi n o a ci d m i x t u r e (A me rs ha m England) an d rat live r a m i n o a c y l - t R N A s yn t h e ta s e s p r e pa r a t i o n (E.C. 6 . 1 . 1 V T r a n s l a t i o n te st w as p e r f o r m e d as d e s c r i b e d b e f or e [7]. D N - a s e I d i g e s t i o n a ss a y w as c a r r i e d ou t in 50 m M T r i s/ H Cl b uf f er pH 7.5 c o n t ai n i n g 4 m M m g 2 + , 1 un it of the e nz y me an d 30 yg of D N A or an e q u i v al e n t a mo u nt of the N A C - D N A c o m p l e x pe r ml. Th e c o m p l e x e s w er e f or m ed as d e s c r i b e d ab o ve w i t h D N A p r e v i o u s l y p u -r i f i ed b y p h en o l e xt-ra ct ion . T h e s am pl e s w e r e i n cu b a t ed at 20°C a nd the c o ur s e of the r e a c t io n w a s f o l l o we d for t w o h o u rs by the a b s o r ba n c e m e a s u r e m e n t s at 260 nm.

D N -a s e S.^ di gestion assay w a s performed according to V o g t

[14] as desc rib ed by P u l k r a b e k [10] usi ng 0.28 m g

of the control D N A or the N A C - D N A complex and 0.0 02 I.U. of the e n zy m e p er ml. Pa pe r c h r o m a t o g r a p h y of th e

[

14

c]

RESULTS AND DISCUSSION

E f f e c t of N AC on t R N A fu nc tio n. In o rd er to c h e c k w h e t h e r i r -r e v e -r s i b l e b i n d i n g of N A C to t R NA im pa r is its fun ctio n, N A C - t R N A c o m p l e x c o n t a i n i n g o n a v er a ge t wo d r u g m o l e c u l e s p er R N A c ha i n w a s f o r m e d in t he p r e s e n c e of d i t h i o t h r e i t o l a n d a s s a y e d for ami- n o ac y l a c c e p t i n g a c t i v i t y a n d its a b i l i t y to m a i n t a i n t r a n s l a -t i o n p ro ce ss. W h e n th e d r u g - t R N A c o m p l e x w as u se d in am in o ac y l- -t R NA s y n t h es i s s y s t e m th e a mo u n t of r a d i oa c t i v e a mi n o acid s b o u n d to t R N A w a s 58% of t he c o n t r o l (Tab. 1). T a b l e 1 Aminoacylation of the NAC-tRNA complex

Aminoacylacja tRNA związanego z NAC

Spécification CPMa % control

tRNA control (20 pg) 638 ± 72 100

NAC-tRNA (20 JJg) 373 ± 52 58 ± 8

tRNA control (10 pg) 472 ± 22 74 ± 3

NAC-tRNA (10 u g )

a CPM • counts per minute.

O n th e o th e r hand, w h e n [1 4C] a m i n o a c y l - t R N A p r e f o r m e d e it he r w i t h t he c o n t r o l or N A C - t r e a t e d t R N A w e r e a d d e d to the p r o t e i n s y n t h e s i s in v i t r o s y s t e m n o d i f f e r e n c e s in th e a m in o a ci d i n c o r -p o r a t i o n t o the a c i d i n s o l u b l e m a t e r i a l w e r e observed' (not shown). H e n c e t he d r u g i n h i b it s a m in o a c id b i n d i n g to t RN A to so me e xt en t w h i l e it d o e s n ot s e e m to a f f e c t t he f u r th e r st ep s of p r o te i n sy nt h es i s. H o we v e r th e i n h i b i t i o n of a m i n o a c y l a t i o n is r e l a t i -v e l y l ow w h e n c o m p a r e d to the d r u g e f fe c t on t he t r a ns c r i p t i o n a l t e m p l a t e a c t i v i t y of DNA. R N A s y n t h es i s on D N A b e a r i n g c om p a r ab l e a m o u n t of N A C m o l e c u l e s c o v a l e n t l y b o u n d (i.e. 20 30 d r u g m o l e c u -les p er 1 0 3 of n uc l e o t i de s ) is r e d u c e d to f ew p e r ce n t [11, 13] . A r e l a t i v e i n s e n s i t i v i t y of a m i n o a c y l a t i o n m a y be d u e to the NAC p r e f e r e n c e to g u a n y l r e s i d u e s s h ow n e i t h e r w i t h s y n t h e ti c p o l y r i -b o n u c l e o t i d e s or p o l y d e o x y n u c l e o t i d e s [3]. T h e r e a re s ome g ur ny l m o i e t i e s in t he h e l ic a l p ar t of a mi n o a ci d a c c e p t i n g ar m w h i c h is p r o b a b l y r e s p o n s i b l e for b i n d i n g of t R N A - s y n t h e t a s e [1, 9]. Ami- n oa c y l r e s id u e s ar e h o w e v e r c o u p l e d to C C A s e q u e nc e s w h i c h o b v i o -u s l y do n ot c o n t a i n g-ua ni ne . A lo w e f f e c t of t h e d r u g on th e t RNA

f un c ti on s p r e s en t e d her e an d a h ig h i n hi b it i on of R NA sy nt hes is in v it r o [5, 12, 13] is in g o o d a gr e em e nt w i t h the o bs e r v a ti o n s m ad e in v i vo i n di c at i ng th at the d ru g in hib it s R N A s yn t he s is to a h ig h er e xt en t t h an p r o t e i n s yn t h es i s [2].

S e n s i t i v i t y of N A C - D N A c o m p l e x to nu cle ases . B i nd i n g of N AC to D NA d e c r e as e s b ot h the rate a nd the e xt e nt of h yd r o l ys i s of the c o m p l e x w i t h p a n c r ea t i c D N- a se I (Fig. 1) an d D N- a se S 1 from A s p e r g i ll u s o ry z ae (Fig. 2).

Incubation time ( min )

Fig. 1. Time course of DNA and NAC-DNA digestion by DN-ase I • — • control DNA (1), O — O NAC-DNA complex (2)

Rys. 1. Trawienie DNA wolnego i związanego z NAC przez DN-azę I 1 - DNA kontrolne, 2 - DNA związane z NAC

Incubation time ( min )

Fig. 2. Time course of DNA and NAC-DNA digestion by DN-ase Sj

® — • c o n t r o l native DNA (1), B — B control heat-denatured DNA (2), O O NAC- -DNA complex (3), □ — O h e a t- d e n at u r e d NAC-DNA complex (A)

Rys. 2. Trawienie DNA wolnego i związanego z NAC przez DN-azę Sj

1 - natywne DNA kontrolne, 2 - kontrolne DNA denaturowane termicznie, 3 - DNA związane z NAC, 4 - DNA związany z NAC denaturowany termicznie

L o w e r seJhsitivity of the c o m p l e x (eith er n o n - h e a t e d or after t h e r m a l d e n a t u r a t i on ) to the l at te r e n z y m e is of int eres t. D N- as e s ho ws a s p e c i f i c i t y to s i n g l e - s t r a n d e d DNA. L o w a f f i n i t y of t he e n z y m e to n a t i v e D N A in th e c o m p l e x i n d i c at e s t h at no si ng l e- - s t r a n d r e gi o n s a pp e a r in D N A u p o n the d r u g bin di ng . Th e time c o u r s e c u r v e of t he r e a c t i o n of d e n a t u r e d N A C - D N A c o m p l e x w it h D N - a s e fa ll s w e l l b e l o w the c o r r e s p o n d i n g c u r v e o b t a i n e d w it h t he h e a t e d c o n t ro l D N A (Fig. 2). T h i s r e su l t is c o n s i s t e n t w i t h ou r p r e v i o u s o b s e r v a t i o n s [3] t ha t i n t er s t r a n d li nk s are in trod

u-Migration distança . ( cm )

Fig. 3. Paper chromatography of [ ^ C ] NAC-RNA complex after its acid hydrolysis Rys. 3. Chromatografia bibułowa produktów chemicznej hydrolizy RNA związanego z

[U C ] NAC

c e d to D N A u p o n the a d duc t fo rma ti on . T h e e x i s t e n c e of the c ro ss- -l ink s in t he c o m p l e x e n a bl e s a p a r t i a l r e c on s t i t u t i o n of the d o u b l e - s t r a n d e d s t ru c tu r es in h e a t d e n a t u r e d D N A he n ce d e c r e a si n g its s e n s i t i v i t y to the s i n g l e - s t r a n d e d s p e c if i c n u c l ea s e

C h e m i c a l h y dr o l y s i s of N A C - p o l y n u c l e o t i d e com pl exe s. C om p le x es c.f N AC w i t h D N A or R N A e x hi b i t r e l a t i v e l y h i g h r e s i st a n c e in

va-ri ou s c o n d i t i o n ^ p a r t i c u l a r l y w h e n fo r m e d at th e hi g h dr ug p o -l y n u c-l e o t i d e m o -l a r ratio. W h e n [14 C] N A C - D N A or R NA c om p le x e s c o n t a i n i n g a b o ut 10- 20 d r u g m o l e c u l e s p er 1 0 3 of n u c l e o t i d es w e re h y d r o l y z e d w i t h 0.3 M H C 1 for 1 h at 100°C, a nd t h en s u b j e ct e d to p ap e r c h r o m a t o g r a p h y w i t h s e v er a l so l ve n ts la rg e a mo u nt of r a -d i o a c t i v i t y r e m a i n e -d at th e o r i g i n (not shown). T he h yd r o ly z a - tes of th e [14 C] NA C- D NA , RNA, or s y n th e t i c p o l y n u c l e o t i d e c o m -p l e x f o r m e d at the lo w er r a ti o n s r e s u l t i n g in b i n d i n g of 0. 2-0. 8 'mole cule s p er 1 0 3 of n u c l e o t i d e s are r e s ol v e d into th ree r a d i o

-a c t i v e sp o ts w h i c h a re d i f f e r e n t f r om the f ree d r u g (Fig. 3). T wo o f t h e m m i g r a t e a nd t he t hi r d on e c o n t a i n i n g a bo u t 20% of the r a -d i o a c t i v i t y r e m ai n s at the or ig in . " It c a n be c o n c l u d e d f ro m the r e su l ts p r e s e n t e d h er e t hat at le a st t hr e e d i f f e r e n t ty p es of the a dd u c t s ar e f o rm e d at the r e -l at i ve q u a n t i t i e s d e p e n d i n g on th e d r u g - p o -l y n u c -l e o t i d e ratio. The p r e s e n t o b s e r v a t i o n s a re c o n s i s t e n t w i t h o ur e a r l i e r r esu lt s w h i c h p r e d i c t e d d i f f e r e n t m o d e s of N A C b i n d i n g to D NA d e p e n d i n g on th e d r u g c o n c e n t r a t i o n [ l l ] . ( ACKNOWLEDGEMENT

We a p p r e c i a t e the r e a d i n g of the m a n u s c r i p t b y E. Sz adow ska. M.A, a n d th e c a p a b l e t e ch n i c a l a s si s t a n c e of M r s M. Fras unek .

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Department of General Chemistry Institute of Physiology and Biochemistry Medical Academy of Łódź

Department of General and Organic Chemistry Institute of Chemistry Medical Academy of Białystok

Ewa Ciesielska, Mariola Piestrzeniewicz, Władysław Gałasiński, Marek Gniazdowski

WŁAŚCIWOŚCI KOMPLEKSÓW NITRAKRYNY Z KWASAMI NUKLEINOWYMI W WYBRANYCH UKŁADACH ENZYMATYCZNYCH

Przebadano niektóre właściwości kompleksów leku przeciwnowotworowego, ni- trakryny ( l-nitro-9-(3,3-N,N-dimetyloaminopropyloamino) akrydyna, Ledakrin, C- -283, NAC) z kwasami nukleinowymi w wybranych układach enzymatycznych

zależ-nych od DNA lub RNA. Kompleksy NAC-DNA charakteryzują się obniżoną aktywno-ścią matrycową w układzie syntezy RNA in vitro. Wykazują one pewną oporność na działanie DN-azy X i DN-azy Sj i na hydrolizę chemiczną. Wiązanie NAC do tRNA obniża jego aminoacyXację. Z drugiej strony, taki aminoacylo-tRNA wykazu-je niezmienioną aktywność w układzie syntezy białka in vitro. W niniejszym artykule przedstawiono również wstępne wyniki chromatografii zhydrolizowanych kompleksów.